The present invention relates to the preparation of a single active protein from a novel fusion gene for use as a reporter gene system. More specifically, this invention relates to the fusion of a luxA and luxB gene from Vibrio harveyi so as to produce a fused lux AB gene which is useful as a reporter gene. The fusion gene encodes a single active fusion protein as its gene product, which is useful as a reporter protein.
It is generally recognized that gene expression studies will benefit from the use of a reporter gene. An ideal reporter gene is easily assayed, both in vivo and in vitro, and is able to indicate the temporal and spatial aspects of gene expression. Optimally, the reporter gene encodes a gene product whose activity is not normally found in the organism of interest and thus may be easily assayed.
A problem of considerable importance in genetic engineering technology is the difficulty of obtaining a promotor sequence that promotes the expression of genes in a host organism. A reporter gene may be fused to a promoter of intersest and the amount of reporter gene product produced is indicative of the relative activity of the promoter. A good example of such a reporter gene is .beta.-galactosidase, an enzyme encoded by the lac Z gene of E. coli. The presence of the lac Z gene product in a cell can be qualitatively determined in whole cells and can be quantitatively measured in cell-free extracts.
An ideal candidate for a reporter gene is the luxA and luxB luciferase gene system from Vibrio harveyi. The gene product of the luxA gene is the .alpha. subunit of the enzyme and the gene product of the luxB gene is the .beta. submit of the enzyme. The .alpha.- and .beta.-subunits encoded by the luxA and luxB genes respectively, form a functionally active heterodimer protein enzyme complex which in the presence of substrates will emit light.
Bacterial luciferase, as exemplified by luciferase derived from Vibrio harveyi (EC 1.14.14.3, alkanol reduced-FMN-oxygen oxidoreductase; 1-hydroxylating, luminescing), is a mixed function oxidase, formed by the association of two different protein subunits .alpha. and .beta.. The .alpha.-subunit has an apparent molecular weight of .about.42,000 d. and the .beta.-subunit has an apparent molecular weight of .about.37,000 d. Cohn et al., 1983, Proc. Acad. Sci. USA 80: 102-123. The .alpha.- and .beta.-subunits must associate to form a 2-chain complex in order to generate functional luciferase enzyme, which catalyzes the light emitting reaction of naturally bioluminescent bacteria, such as Vibrio harveyi (U.S. Pat. No. 4,581,335; Belas et al. 1982, Science 218:791-793), Vibrio fischeri (Engebracht et al., 1983, Cell 32:773-781; Engebrecht and Silverman, 1984, Proc. Natl. Acad. Sci. U.S.A. 81:4154-4158) and other marine bacteria. Specifically, bacterial luciferase catalyzes the flavin-mediated hydroxylation of a long-chain aldehyde to yield carboxylic acid and an excited flavin; the flavin decays to ground state with the concomitant emission of blue green light (.lambda.max=490 nm). Legocki et al., 1986, Proc. Nat. Acad. Sci. USA 81:9080.
The reaction in vitro is initiated by injection of reduced flavin mononucleotide (FMNH.sub.2) into a vial containing bacterial luciferase, oxygen, and a long-chain aldehyde, usually n-decyl aldehyde. U.S. Pat. No. 4,581,335. The reaction pathway is as follows: ##STR1##
The number of photons produced in the reaction is proportional to the amount of protein enzyme present when substrates are in excess. Since flavin mononucleotide (FMN) is a normal constituent of most cells and n-decyl aldehyde freely crosses membranes, the product of the luxA and lux B genes can be quantitatively measured in intact FMN-containing cells.
A different luciferase enzyme system is found in the firefly, Photinus pyradis. Firefly luciferase (EC 1.13.12.7, luciferin: oxygen 4-oxidoreductase; decarboxylating, ATP hydrolysing) has an apparent molecular weight of .about.62,000 d. and requires luciferin, ATP, and O.sub.2 as substrates. Specifically, firefly luciferase catalyzes the light producing, adenosine triphosphate (ATP)-dependent oxidation of luciferin. The reaction pathway catalyzed by firefly luciferase is as follows: ##STR2## Thus, firefly luciferase has an entirely different gene structure, different protein structure, different enzyme activity and reaction pathway as compared with a bacterial luciferase. A required cofactor in this reaction pathway is luciferin, which is a compound that is rarely present in cells other than firefly cells. Since luciferin does not readily cross membranes, it is difficult to use the firefly luciferase system as a reporter system in intact cells. The firefly luciferase gene has been cloned and sequenced (deWet et al., 1985, Proc. Natl. Acad. Sci. 82:7870-7873; deWet et al., 1987, Mol. Cell. Biol. 7:725-737).
The luxA and luxB genes of Vibrio harveyi have been cloned (Belas et al., 1982, Science 218: 791-793; Cohn et al. 1983, Proc. Natl. Acad. Sci. USA 80: 120-123) and sequenced (Cohn et al., 1985, J. Biol. Chem. 260: 6139-6146; Johnson et al., 1986, J. Biol. Chem. 261: 4805-4811). The luxA and luxB genes of Vibrio harveyi have been used as reporters of gene expression in a filamentous bacterium Streptomyces coelicolor (Schauer et al., 1988, Science 240:768-772). One disadvantage to the use of the Vibrio harveyi luciferase 2-gene system as a reporter gene system is that it is expressed in bacteria as a polycistronic message from a single promoter. Since eukaryotic cells do not have analogous structures for expression, the use of the Vibrio harveyi luciferase gene system in eukaryotic cells is limited. One solution which was utilized in the plant system, so as to overcome this disadvantage, was to fuse a plant promoter to each of the luxA and luxB genes from Vibrio harveyi to form two separate transcription-translation cassettes. See Koncz et al., 1987, Proc. Natl. Acad. Sci. USA 84:131. These two cassettes (cassette 1=T.sub.R -DNA gene 1' promoter luxA; cassette 2=T.sub.R -DNA gene 2' promoter luxB) containing the luxA and luxB structural genes from Vibrio harveyi were introduced into a plant expression vector and transferred into tobacco and carrot cells by Agrobacterium-mediated or direct DNA transformation. Simultaneous expression of the luxA and luxB gene products was monitored by protein immunoblot analysis. Luciferase-mediated light emission in an in vitro assay provided evidence of the assembly of the two protein subunits into a functional dimeric enzyme in plant protoplasts, in transformed calli, and in leaves of transformed plants. However, this method is laborious and is not always feasible due to restrictions imposed by the required plasmid construction.
Several other reporter genes such as .beta.-galactosidase, chloramphenicol acetyltransferase (CAT) and firefly luciferase have been described but may not be advantageous because of their assays for activity or because of difficulties in construction or expression in certain cell types. In particular, the use of firefly luciferase as an in vivo marker is complicated by toxicity and variable access of its substrate to eukaryotic compartments (Ow et al., 1986, Science 234:856-859; deWet et al., 1985, supra). These reporter genes are summarized as follows: .beta.-galactosidase (MacGregor et al., 1987, Somatic Cell Mol. Genet. 13:253-266); galactokinase (e.g., Rosenberg et al., 1983, Science 222:734-739; McKenney et al., 1981, in Gene Amplification and Analysis, Volume 2, pp. 383-415, Elsevier/North-Holland, New York); Murooka and Mitani, 1985, J. Biotechnol. 2:303-316; .beta.-glucuronidase (e.g., Jefferson et al., 1986, Proc. Natl. Acad. Sci. 83:8447-8541); human growth hormone (e.g., Seldon et al., 1986, Mol. Cell. Biol. 6:3173-3179); chloramphenicol acetyltransferase (CAT) (e.g., Tsukada, et al., 1987, J. Biol. Chem. 262:8743-8747; Carbonell and Miller, 1987, Appl. Environ. Microbiol. 53:1412-1417; Boulet et al., 1986, Proc. Natl. Acad. Sci. USA 83:3599-3603; Jameson et al., 1986, Endocrinology 119:2560-2567; Montminy et al., 1986, Proc. Natl. Acad. Sci. USA 83:6682-6686); Tn5 neomycin phosphotransferase (e.g., Kaulen et al., 1986, EMBO J. 5:1-8; Simpson et al., 1985, EMBO J. 4:2723-2730) and firefly luciferase (e.g., Ow et al., 1987, Proc. Nat. Acad. Sci. USA 84:4870-4874, Ow et al., 1986, Science 234:856-859).
The present invention represents a significant and distinct contribution to the art in that is provides a means for utilizing the luxA and luxB genes from Vibrio harveyi as a reporter gene in a variety of different cell types. The luxA and luxB genes from Vibrio harveyi are fused so as to produce a novel fusion gene which encodes single active protein product. Because of the perceived requirement for physical interaction of the .alpha.- and .beta.-subunits encoded by the luxA and luxB genes, respectively, in order to generate a functional luciferase enzyme complex having light-producing activity, it was unexpected that a fusion protein which combined the amino acid sequences of the .alpha.- and .beta.-subunits on a single polypeptide chain would be functionally active. However, a luxAB fusion gene according to the present invention, when expressed as a fusion protein, surprisingly produces a novel luciferase enzyme that is functionally active both in vitro and in vivo. A fusion protein according to the present invention is a simple and extremely sensitive gene reporter. The luxAB gene of the present invention has been cloned with a variety of expression vectors, suitable for use in bacterial cells, yeast cells, plant cells, animal cells and fungal cells. This protein is an ideal reporter of gene expression in a wide variety of host cells. The activity can be measured in intact, living cells which contain FMNH.sub.2, when the volatile substrate decanal is provided. Since many cells do contain FMNH.sub.2, a reporter system that comprises a luxAB fusion gene of the present invention allows real-time measurement of gene expression. Because only a single protein (encoded by a single transcriptional unit) need be expressed, utilization of this reporter system in eukaryotic cells is greatly facilitated.
For purposes of the present invention as disclosed and claimed herein, the following terms are as defined below.
.alpha. subunit--a polypeptide chain encoded by a luxA gene, which forms an enzyme complex with a .beta. subunit, the enzyme complex having luciferase activity.
.beta. subunit--a polypeptide chain encoded by a luxB gene, which forms an enzyme complex with a .alpha. subunit, the enzyme complex having luciferase activity.
Fusion Gene--a DNA sequence comprising two or more genetic sequences (genes or portion of genes) operably linked to one another.
Fusion Protein--a protein encoded by a fusion gene.
luxA--a gene encoding an .alpha.-subunit of a bacterial luciferase.
luxB--a gene encoding a .beta.-subunit of a bacterial luciferase.
ori--a plasmid origin of replication.
PGK--the transcriptional and translational activating sequence of the yeast Saccharomyces cerevisiae phosphoglycerate kinase gene.
phage--a bacterial virus, also referred to as bacteriophage.
plasmid--an autonomous self-replicating extrachromosomal circular DNA.
Recombinant DNA Cloning Vector--any autonomously replicating agent, including but not limited to plasmids, containing a DNA molecule to which one or more additional DNA segments can or have been added.
Recombinant DNA Expression Vector--any recombinant DNA cloning vector into which one or more transcriptional and translational activator sequence(s) have been incorporated.
Reporter Gene--any genetic material, whose utilization by the transcriptional and/or translational apparatus derived from a cell (e.g., intact cell or cell-free extract) can be monitored, for example, any DNA sequence that is fused to a promoter sequence of interest so as to measure the relative activity of the promoter sequence.
Reporter Protein--a protein encoded by a reporter gene.
Restriction Fragment--any linear DNA molecule generated by the action of one or more restriction enzymes.
Transfectant--a recipient host cell that has undergone transfection.
Transfection--the introduction of DNA into an animal host cell, such as COS-1 cells (may also be termed transformation).
Transformant--a recipient host cell that has undergone transformation.
Transformation--the introduction of DNA into a recipient host cell.